Recent development in the field of pericentromeric heterochromatin formation and X-inactivation revealed some striking similarities between the two processes. X-inactivation in mammalian female embryonic stem cells can be regarded a developmentally regulated facultative heterochromatin formation process. The long- range goal of this project is to contribute to the understanding of Xist RNA initiated selective repression of inactive X chromosome. Specific aims are to: 1) identify and study the effector proteins that interact with Xi- specific histone modifications, and 2) elucidate the mechanism by which Xist RNA recruit Ezh2 and possible other HMTs to participate in X inactivation. A series of biochemical, biophysical, immunocytological, and cell biological approaches have been proposed, first to examine Cbx proteins for possible interactions with histone modifications and RNAs, then to expand to more potential candidates and eventually perform unbiased screen for more key players in X-inactivation process. The proposed research will not only further our understanding of roles played by heterochromatin as a widely-used epigenetic regulator, but also provide insights into an important aspect of early embryonic development and embryonic stem cell differentiation. [unreadable] [unreadable] [unreadable]